JCB: Report Ovastacin, a cortical granule protease, cleaves ZP2 in the zona pellucida to prevent polyspermy Anna D. Burkart, Bo Xiong, Boris Baibakov, Maria Jiménez-Movilla, and Jurrien Dean Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892 he mouse zona pellucida is composed of three gly- before, but not after, fertilization. Recombinant ovasta- coproteins (ZP1, ZP2, and ZP3), of which ZP2 is cin cleaved ZP2 in native zonae pellucidae, documenting T proteolytically cleaved after gamete fusion to pre- that ZP2 was a direct substrate of this metalloendopro- vent polyspermy. This cleavage is associated with exo- tease. Female mice lacking ovastacin did not cleave ZP2 cytosis of cortical granules that are peripherally located after fertilization, and mouse sperm bound as well to subcellular organelles unique to ovulated eggs. Based Astl-null two-cell embryos as they did to normal eggs. on the cleavage site of ZP2, ovastacin was selected as Ovastacin is a pioneer component of mouse cortical a candidate protease. Encoded by the single-copy Astl granules and plays a definitive role in the postfertiliza- gene, ovastacin is an oocyte-specific member of the tion block to sperm binding that ensures monospermic astacin family of metalloendoproteases. Using specific fertilization and successful development. antiserum, ovastacin was detected in cortical granules Introduction Because polyspermy is an embryonic lethal, at least three post- Deng et al., 2003). Cortical granules become competent to fertilization blocks to gamete interactions have evolved in mice. undergo exocytosis just before ovulation, and the 8,000 cortical The first two occur rapidly after fertilization and prevent addi- granules observed in fully grown oocytes decline to 4,800 tional sperm from fusing with the egg’s plasma membrane or in ovulated eggs (Ducibella et al., 1994). Fertilization triggers penetrating the extracellular zona pellucida surrounding eggs cortical granule migration to the plasma membrane, where they and preimplantation embryos (Sato, 1979; Stewart-Savage and fuse and exocytose their contents (Wessel et al., 2001; Ducibella Bavister, 1988). The third and definitive block occurs over et al., 2002). several hours and ensures that sperm do not bind to the surface Little is known about the contents of mouse cortical gran- of the zona pellucida (Inoue and Wolf, 1975; Baibakov et al., ules (Liu, 2011), and the only documented biological function is 2007). The molecular basis of the first two blocks remains the postfertilization cleavage of ZP2 (Bleil et al., 1981), which, THE JOURNAL OF CELL BIOLOGY largely unknown, and the third correlates with egg cortical granule along with ZP1 and ZP3, forms a structured extracellular glyco- exocytosis (Barros and Yanagimachi, 1971). matrix that surrounds mouse eggs (Bleil and Wassarman, 1980). Cortical granules are Golgi apparatus–derived, membrane- Cleavage of ZP2 is N terminal of a diacidic residue (Gahlay bound vesicles (0.2–0.6 µm) that accumulate during oogenesis et al., 2010), a known cleavage site for the astacin family of and form a uniform layer in the cortex of fully grown mouse metalloendoproteases. Ovastacin (Astl, the official gene name) eggs. The observed 15-fold increase in cortical granules during is expressed in growing mouse oocytes and has a signal peptide oocyte growth reflects both an increase in granule density to direct it into a secretory pathway but has no known function and in the cortical area as oocytes increase in diameter from (Quesada et al., 2004). We now localize ovastacin as a pioneer 40 to >80 µm (Zamboni, 1970; Nicosia et al., 1977; Ducibella component of mouse egg cortical granules and document its et al., 1994). During meiotic maturation and germinal vesicle ability to modify the zona pellucida to prevent postfertilization breakdown, cortical granules redistribute and are excluded sperm binding and provide a definitive block to polyspermy. from the region of the metaphase I spindle (Ducibella et al., 1988a; This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www Correspondence to Jurrien Dean: [email protected] .rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http:// Abbreviations used in this paper: ES, embryonic stem; PVP, polyvinylpyrrolidone. creativecommons.org/licenses/by-nc-sa/3.0/). The Rockefeller University Press $30.00 J. Cell Biol. Vol. 197 No. 1 37–44 www.jcb.org/cgi/doi/10.1083/jcb.201112094 JCB 37 Figure 1. Localization of ovastacin. (A) Mouse ZP2 (713 aa) is proteolytically processed by cleavage of a signal peptide (Sig) and at a bibasic motif (blue arrow) upstream of a transmembrane (TM) domain to release the ectodomain (ZP235-633) that participates in the extracellular zona pellucida. After fertiliza- tion, ZP2 is cleaved upstream of a diacidic motif (169DE170). Conserved cysteine residues (yellow vertical lines) and potential N-linked glycosylation sites (red circles) are indicated. (B) The diacidic (shaded) proteolytic cleavage site (arrow) in the N-terminal region of mouse ZP2 is conserved in human, bonnet monkey, cow, pig, dog, cat, rabbit, hamster, and rat. (C) A schematic of mouse ovastacin including a signal peptide, a Zn+2-binding (blue histidine residues) enzyme-active site (red glutamic acid with an asterisk), and the antigen (395PLALF- PEARDKPAP408) used to generate a rabbit pAb. Cysteine residues and potential glycosylation sites are indicated as in A. (D) Unfertilized eggs and one-cell embryos from normal mice were imaged by confocal microscopy and differential interference contrast (DIC) after staining with rabbit anti-ovastacin (Ovst) anti- body, LCA-FITC (a marker of cortical granules), and the nuclear stain Hoechst 33342. (E) An immunoblot of lysates from unfertilized eggs (150) and two-cell embryos (150) was probed with antibody to ovastacin. Results and discussion conclude that ovastacin is expressed in eggs, where it localizes to peripheral cortical granules and is discharged during post- Ovastacin is present in mouse egg fertilization cortical granule exocytosis. cortical granules ZP1, ZP2, and ZP3 form the extracellular zona pellucida that In the absence of ovastacin, cortical surrounds mouse eggs and early embryos (Bleil and Wassarman, granules persist in ovulated eggs 1980). Sperm bind to eggs but not two-cell embryos, and the To determine its function, the single-copy Astl gene was suc- only documented biochemical change in the zona matrix is cessfully targeted for ablation in mouse embryonic stem (ES) cleavage of ZP2 (Fig. 1 A; Bleil et al., 1981). This cleavage is cells using a neomycin cassette flanked 5 and 3 by 5.3 and associated with cortical granule exocytosis and is N terminal 1.5 kbp of homology, respectively (Fig. 2 A). Colonies were of a diacidic motif, 168DE169 (Gahlay et al., 2010). The site is initially screened by PCR, and 14 positive clones were confirmed well conserved among mammals (Hasegawa et al., 1994; Tian by Southern blot analysis using 5 and 3 probes outside the et al., 1999; Lindsay and Hedrick, 2004), but the identity of regions of homology (Fig. 2 B). After blastocyst injection, two the presumptive cortical granule protease has as of yet remained coat-color chimeric male mice were identified, and germline unknown (Fig. 1 B). Ovastacin (Fig. 1 C) is a member of the transmission of the null allele was confirmed by the genotype large astacin family of metalloendoproteases (Dumermuth et al., of tail DNA (Fig. 2 C). Mice, bred to homozygosity for the 1991; Bond and Beynon, 1995), which cleave upstream of mutant Astl allele, were fertile. Although there was a modest diacidic residues. Because of its restricted expression in mouse decrease in fecundity, there was considerable overlap in the size oocytes (Quesada et al., 2004), ovastacin was selected as a of liters (Fig. 2 D), which may reflect effects of mixed genetic candidate protease for the postfertilization cleavage of ZP2. backgrounds. Taking advantage of the unique C-terminal extension of To confirm the absence of ovastacin protein, eggs and ovastacin, a peptide-specific 395( PLALFPEARDKPAP408) rabbit two-cell embryos were stained with LCA-FITC or ovastacin antibody was used to image ovulated eggs by confocal micros- antibodies and imaged by confocal microscopy. Colocalization copy. LCA-FITC (Lens culinaris agglutinin conjugated to FITC) in the periphery of eggs, but not two-cell embryos, was ob- is a marker of cortical granules (Ducibella et al., 1988b), and its served in normal mice (Fig. 3 A). Similar results were obtained colocalization in the periphery of ovulated eggs indicates the for eggs and embryos from heterozygous null females, although presence of ovastacin within these granules (Fig. 1 D). Disap- the intensity of the signals was diminished. As anticipated, pearance of ovastacin after fertilization and cortical granule ovastacin was not detected in the homozygous null eggs, but, exocytosis was observed by confocal microscopy (Fig. 1 D) and unexpectedly, LCA-FITC reactivity was lost as well (Fig. 3 A). confirmed by an immunoblot that detected the two known To determine whether this reflected an absence of cortical gran- isoforms of the enzyme (Quesada et al., 2004) in eggs but not ules, AstlNull eggs were stained with WGA. Because WGA strongly two-cell embryos (Fig. 1 E). From these observations, we reacts with the zona pellucida, it is difficult to detect peripheral 38 JCB • VOLUME 197 • NUMBER 1 • 2012 Figure 2. Establishment of AstlNull mice. (A) A schematic of the normal and AstlNull alleles after targeting with a construct containing positive (phosphoglucokinase [PGK]-Neo) and negative (MC1-TK) selectable markers. The thicker lines represent the 5.3- and 1.5-kbp homologous arms that are 5 and 3, respectively, to the Neo cas- sette.